Fermentaive acetylation of steroidal 3beta-ols

ABSTRACT

A PROCESS FOR ACETYLATING STEROIDAL 3B-OLS BY FERMENTATION WITH PENICILLIN A.T.C.C. 16501 OR SPICARIA 20152 OR 20153 IS DISCLOSED.

United States Patent 3,597,320 FERMENTATIVE ACETYLA'HON 0F STEROIDAL 3/:I-OLS Stephen Kraychy, Northbrook, and Seth S. Mizuba, Morton Grove, 1111., assignors to G. D. Searle 8: (30., Chicago, iill.

No Drawing. Continuation-impart of application Ser. No. 575,246, Aug. 26, 1966. This application Dec. 11, 1968, Ser. No. 783,127

lint. Cl. C07c 167/28 US. Cl. 195-51 12 Claims ABSTRACT OF THE DISCLOSURE A process for acetylating steroidal 3fi-ols by fermentation with Penicillium A.T.C.C. 16501 or Spicaria 20152 or 20153 is disclosed.

The application for Letters Patent securing the invention herein described and claimed is a continuation-inpart of the applicants copending application Ser. No. 575,246 filed Aug. 26, 1966.

This invention relates to fermentative acetylation of steroidal 3/3-ols. More particularly, this invention provides a process whereby a steroid containing a SIB-hydroxy substituent is converted to the corresponding 35- acetoxy compound via the esterifying activity of selected species of Penicillium and Spicaria. The particular species of Penicillium and Spicaria referred to are those identified by the American Type Culture Collection code numbers 16501, 20152, and 20153, each of which the collection agency is empowered to make available at any time upon payment of their usual fee.

Contrary to what might be expected, the presence of other substituents in the steroid molecule, including but not limited to 115 and/or 17 hydroxyls, does not appear to interfere with 3-acetylation. The only known exceptions to this generalization are 11cc or 70: or B hydroxyls which, inexplicably, render the process inoperable.

Typical steroidal 35-015 adapted to the fermentative acetylation herein described and claimed are 3/3-hydroxyandrost--en-17-one, 3fl-hydroxyandrostan-17-one, 3 8- hydroxypregn-S-en-20-one, androst-S-ene-BB,17-diol, 17- (lower alkyl)androst-5-ene-3/3,17diol, androst-S-ene-Bfl- 1 15,17-triol, and 3-(313,17-dihydroxyandrost-5-en-17-yl)- propionic acid and corresponding 'y-lactone.

Fermentation is ordinarily carried out in a growing culture of any of the organisms above identified, to which the steroid to be esterified is added during the incubation period. Alternatively, the steroid can be incorporated in the nutrient medium prior to inoculation. A preferred but a critical range of concentrations of the steroid in the culture is 0.0025 to 0.1%.

The time required for completion of the acetylation varies widely, with fermentations of from 2 to 96 hours duration being representative.

Assimilable nitrogen and carbon should of course be present during fermentation for optimum results; and an adequate supply of sterile air should be maintained, for example by exposing a large surface of the medium to the air or by passing it through the medium in quantities sufficient to support submerged growth.

Nitrogen sources are commonly those normally employed for fermentations, including naturally-occurring organic materials such as soy bean meal, corn steep liquor, meat extract, peptone, and/or distillers solubles, as well as synthetic nitrates and/or ammonium compounds. Among the foregoing materials, meat extract :and peptone serve also as carbon sources. Other carboncontaining substances suitable for, and conventionally used as, nutrients are the carbohydrates-for example, glycerol, glucose, fructose, dextrose, sucrose, lactose, maltose, dextrines starches and whey.

The 3-acetates prepared by the instant process are useful both because of their valuable pharmacological properties and the fact that they serve as intermediates to pharmacologically valuable products. Thus, for example, US. 3,109,850 (column 2, lines 12-19) discloses that 3/3-acetoxyandrost-5-ene-l1,17-diols are naturiuretic; and US. 3,159,622 (Example 1) discloses 3-(3fl-acetoxy- 17,8-hydroxyandrost-5-en-17a-yl)propionie acid 'y-lactone as the starting material for preparation of 3-(17,8-hydroxy 3 oxo-6,B-nitroandrost-4-en-17a-yl)propionic acid 'y-lactone, which blocks the effect of desoxycorticosterone acetate on urinary sodium and potassium and inhibits cotyledonous seed germination and the growth of bacteria such as Diplococcus pneumoniae.

The following examples describe in detail various illustrative applications of the process of this invention. However, the invention is not to be construed as limited thereby, either in spirit or in scope, since it will be apparent to those skilled in the fermentative art that many modifications, both of materials and of techniques, may be practiced without departing from the purpose and intent of this disclosure. -In the examples hereinafter set forth, temperatures are given in degrees centigrade and relative amounts of materials in parts by weight, except as otherwise noted. Specific rotations refer to the D line of sodium.

The fermentations in each of the examples hereinafter are carried out as follows: A nutrient medium consisting of 1000 parts of dextrose, 150 parts of cotton seed meal, 6 parts of concentrated hydrochloric acid, parts of corn steep liquor, 5 parts of silicone anti-foam emulsion, and 25,000 parts of water is sterilized by heating to 121, whereupon it is cooled to about 25 and then inoculated with an aqueous suspension of 7-day old spores and mycelium from a culture of the indicated organism. The medium is maintained at about 25 for approximately 24 hours, during which time a stream of sterile air is passed through and the developing culture is agitated to produce submerged growth. A solution of 10 parts of the indicated 35-01 in approximately 200 parts of acetone is then introduced and agitation with aeration at about 25 thereupon resumed for the indicated fermentation time, at which point the mixture is extracted with dichloromethane. The dichloromethane extract is stripped of solvent and the residue worked up as described in the individual examples.

EXAMPLE 1 J CHsOOO-C'J 3 EXAMPLE 2 From an eluate comprising 10% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from aqueous methanol, 3,8-acetoxyandrost- -en-17fi-ol is obtained. The product has the formula set forth in Example 1.

CHaCOO EXAMPLE 3 3/3,17B-diacetoxyandrost-5-ene and 3/3-acetoxyandrost- 5-en-17fi-oL-Upon 72-hour fermentation of androst-S- ene-3fi,l7fl-diol with Spicaria sp. A.T.C.C. 20153 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates Which is chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising 5% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the resultant residue from 20% hexane in ether, 3fi17fl-diacetoxyandrost-S-ene melting at approximately l55-161 is obtained. The product has the formula set forth in the first paragraph of Example 2.

From an eluate comprising ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from aqueous methanol, 3fi-acetoxyandrost- 5-en-l7fl-ol melting at 138149.5 is obtained. The product has the formula set forth in Example 1.

EXAMPLE 4 3fi-acetoxy-17u methylandrost-S-en-l7fi-ol.--Substitution of 17ot-methylandrost-5-ene-36,17/3-diol for the androst-5-ene-3fi,l7B-diol called for in Example 1 affords, by the procedure there set forth, 3fl-acetoxy-17a-methylandrost-5-en-17B-ol, having the formula CHaCOO EXAMPLE 5 3 3-acetoxyandrost-5-ene-l113,17fi-di0l. Upon fermentation for 79 /2 hours of androst-5-ene-3fl,115,17fi-triol with Penicillium sp. A.T.C.C. 16501 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates which is washed with pentane and then chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising 40% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from ethyl acetate, 3B-acetoxyandrost-5-ene-l15,1713- diol melting at approximately 155-156 and having a specific rotation of 43.5 (1% in chloroform) is obtained. The product has the formula CH COO- EXAMPLE 6 3 (Mi-acetoxy 17,8 hydroxyandrost 5 en-17a-yl)- propionic acid 'y-lactone.Substitution of 3-(3fl,17,8-dihydroxyandrost 5 en 17oz yl)propionic acid y-lactone for the androst-5-ene-3p,l7,8-di01 called for in Example 1 affords, by the procedure there set forth, 3-(313- acetoxy 17fl-hydroxyandrost-5-en-l7ot-yl)propionic acid 'y-lactone, having the formula.

EXAMPLE 7 EXAMPLE 8 3 (3/3-acetoxy 17B hydroxyandrost 5 en-17a-yl) propionic acid -lactone.Substitution of Spicaria sp. A.T.C.C. 20153 for the Spicaria sp. A.T.C.C. 20152 called for in Example 7 affords, by the procedure there set forth, S-(SB-acetoxy 17p hydroxyandrost-5-en-l7oayl)propionic acid 'y-lactone. The product has the formula set forth in Example 6.

EXAMPLE 9 3fl-acetoxyandrost 5 en-17-one.-Upon 24-hour fermentation of 3 B-hydroxyandrost-S-en-l7-one with Penicillium sp. A.T.C.C. 16501 in accordance with the procedure described in the last paragraph preceding the examples,a residue eventuates which is washed with pentane and then chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising approximately 20% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from acetonitrile and then from ether, colorless 3,8-acetoxyandrost-5-en-17-0nc melting at 167.5-

169.5 and having a specific rotation of 11 (1% in chloroform) is obtained. The product has the formula CHaCOO- EXAMPLE 3fi-acetoxyandrost 5 en 17 one.Upon fermentation for 28 /2 hours of 3B-hydroxyandrost 5 en-l7-one with Spicaria sp. A.T.C.C. 20152 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates which is taken up in benzene. The benzene solution is chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising ethyl acetate in benzene, 0n evaporation of solvents and recrystallization of the resultant residue from a 1:5 mixture of ethyl acetate and ether, 3/3-acetoxyandrost-5-en-17-one melting at 1705-1725 is obtained. The product has the formula set forth in Example 9.

EXAMPLE 11 3e acetoxyandrost-S-en-17-one. Upon -hour fermentation of 3fi-hydroxyandrost-5-en-17-one with Spicaria sp. A.T.C.C. 20153 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates which is washed with pentane and chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising 5% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from a mixture of 50% hexane in ether, Zfi-acetoxyandrost-S-em17-one melting at 161-166 is obtained. The product has the formula set forth in Example 9.

EXAMPLE 12 3-,8-acetoxypregn 5 en-20-one.Upon fermentation for 22 /2 hours of 3s hydroxypregn-5-en-20 one with Penicillium sp. A.T.C.C. 16501 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates which is taken up in warm ethyl acetate. Benzene is added to the ethyl acetate solution q.s. 85%; and the resultant solution is chromatographed on silica gel, using 85% benzene in ethyl acetate as developing solvent. The eluate is stripped of solvent by vacuum distillation, and the residue is consecutively recrystallized from ethyl acetate and ethanol to give acetoxypregn-5-en-20-one melting at approximately 149.5- l50.5. The product has the formula r 11 0 C=O on o 0 o J EXAMPLE 13 3 5-acetoxypregn 5 en 20 one.--Upon 72-hour fermentation of 3B-hydroxypregn-5-en-20-one with Spicaria sp. A.T.C.C. 20152 in accordancewith the procedure de scribed in the last paragraph preceding the examples, a residue eventuates which is chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising 5% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the re sultant residue from ether, 3e-acetoxypregn-5-en-20-one melting at 144.5-149.5 is obtained. The product has the formula set forth in Example 12.

EXAMPLE 14 3,8-acetoxypregn-5-en-20-one.Substitution of Spicaria sp. A.T.C.C. 20152 in accordance with the procedure decalled for in Example 13 affords, by the procedure there detailed, 3B-acetoxypregm5-en-20-one melting at 143.5- 148.5 The product has the formula set forth in Example 12.

EXAMPLE 15 3fl-acetoxy-5a-androstan-17-one.Upon 49-hour fer mentation of 3 [3 hydroxy 5a androstan 17 one with Penicillium sp. A.T.C.C. 16501 in accordance with the procedure described in the last paragraph preceding the examples, a residue eventuates which is chromatographed on silica gel, using benzene and ethyl acetate as developing solvents. From an eluate comprising approximately 5% ethyl acetate in benzene, on evaporation of solvents and recrystallization of the residue from ethyl acetate, 3,8-acetoxy-5a-androstan-17-one melting at approximately is obtained. The product has the formula CH3COO- What is claimed is:

1. The process of acetylating the 3,8-hydroxyl of a steroid by subjecting it to the esterifying action of an organism selected from the group consisting of Penicillium sp. A.T.C.C. 16501, Spicaria sp. A.T.C.C. 20152, and Spicaria sp. A.T.C.C. 20153, said steroid being selected from the group consisting of 3 fl-ols wherein neither an lla-hydroxyl nor a 7-hydroxyl is present.

2. A process according to claim 11 wherein the organism is Pencillium sp. A.T.C.C. 16501.

3. A process according to claim 1 wherein the organism is Spicaria sp. A.T.C.C. 20152.

4. A process according to claim ll wherein the organism is Spicaria sp. A.T.C.C. 20153.

5. A process according to claim 1 wherein the steroid is selected from the group consisting of androst-5-ene-3B, 17fl-diol, 17et-methylandrost-5-ene-3fi,17,8-diol, androst-S- cue-313,115,17/3-triol, 3-(3,B,17/3-dihydroxyandrost-5 en- 17a-yl)propionic acid 'y-lactone, 3,8-hydroxyandr0st-5-en- 17-one, 3fi-hydroxypregn-5-en-20-one, and 3B hydroxy- 5u-androstan-17-one.

6. A process according to claim 1 in which the steroid is androst-5-ene-3/3,l7B-diol.

7. A process according to claim 1 in which the steroid is 17ot-methylandrost-5-ene-3,8,17B-diol.

8. A process according to claim 1 in which the steroid is androst-5-ene-3B,11B-17fl-triol.

9. A process according to claim 11 in which the steroid is 3-(35,17fl-dihydroxyandrost-5-ene 17cc yl)propionic acid -lactone.

10. A process according to claim 1 in which the steroid is 3,8-hydroxyandrost-5-en-17one.

111. A process according to claim 11 in which the steroid is 3fi-hydroxypregn-S-en-ZO-one.

12. A process according to claim 11 in which the steroid is 3fl-hydroxy-Sot-androstan-l7-one.

References Cited UNITED STATES PATENTS 3,431,173 3/1969 Waard et a1. 51

ALVIN E. TANENHOLTZ, Primary Examiner UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 ,597,32 Dated August 5: 97

In en (s) Stephen Kraychy and Seth S. Mizuba It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 5 "a critical" should be -a.critical--.

Column 2, line 5, "dextrines starches" should be dextrines, starches Column 2, formula,

1: 5 n HBC should be Column 3, line 2, "acetoxy-androst-" should be acetoxyandrost- Column 3, line 39, "361753" should be 55, 1'76 Column 5, line 69, "do" should be de- Column 6, lines 5-6, "20152 in accordance with the procedure de-" should be 20155 for the Spicaria sp. A.T.C.C. 20152 Column 6, line 59, "115-176" should be 11B, 17B

Column 6, line 61, "5-ene-" should be 5-en- Signed and sealed this 21 st day of March 1972.

(SEAL) Attest:

EDWARD M.FLETCHER, JR. ROBERT GO'ITSCHALK Attesting Officer Commissioner of Patents 

